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Fig. 4. Myelin membrane sheet formation is reduced by dystroglycan-blocking
antibodies. (A) Oligodendrocytes were differentiated for 2 or 4
days on PDL or laminin (Lm) in the presence of dystroglycan-blocking
antibodies (anti-DG) or control antibodies. Indirect immunofluorescence was
used to detect myelin basic protein (MBP) expression. The percentage of
MBP-positive cells was determined using DAPI nuclear stain to obtain total
cell counts per field. In the presence of blocking antibodies, no change in
the percentage of MBP-positive cells was observed at either time point or on
either substrate. (B) The average percentage of cells differentiated on
laminin that express galactocerebroside C (GalC) following treatment with
dystroglycan-blocking antibodies (anti-DG) or control antibodies. No
significant change was found. (C) Typical morphology at day 2.
Oligodendrocytes were differentiated for 2 days on laminin in the presence of
dystroglycan-blocking or control antibodies. MBP immunofluorescence was used
to visualize differentiated cells. Nuclei were detected using DAPI nuclear
stain. (D) Typical morphology at day 4. Oligodendrocytes were
differentiated for 4 days in the presence of dystroglycan (DG) or control
antibodies. MBP immunofluorescence was used to visualize differentiated cells.
Nuclei were detected using DAPI nuclear stain. (E) Myelin membrane
sheet complexity is reduced by DG-blocking antibodies. A morphology
classification scheme (bottom) was used to evaluate the degree of myelin
membrane in MBP-positive cells. Examples of MBP-expressing cells: stages 1, 2
and 3 show increasing levels of process outgrowth and branching, without
myelin membrane, whereas stages 4, 5 and 6 show increasing levels of
complexity and myelin membrane. The percentage of cells within each category
is shown for oligodendrocytes differentiated for 2 or 4 days in the presence
or absence of DG-blocking antibodies (control, black squares; anti-DG, gray
triangles). In the presence of DG-blocking antibodies, no difference in
morphology profile is observed at day 2; however, at day 4, a significant
shift away from more-complex myelin membrane structures is observed
(*P<0.05, **P<0.01, error bars
represent s.d.). Scale bars: 100 µm.
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